title: Structural and functional analysis of the yeast N-acetyltransferase Mpr1 involved in oxidative stress tolerance via proline metabolism.
abstract: Mpr1 (sigma1278b gene for proline-analog resistance 1), which was originally isolated as N-acetyltransferase detoxifying the proline analog L-azetidine-2-carboxylate, protects yeast cells from various oxidative stresses. Mpr1 mediates the L-proline and L-arginine metabolism by acetylating L-Δ(1)-pyrroline-5-carboxylate, leading to the L-arginine-dependent production of nitric oxide, which confers oxidative stress tolerance. Mpr1 belongs to the Gcn5-related N-acetyltransferase (GNAT) superfamily, but exhibits poor sequence homology with the GNAT enzymes and unique substrate specificity. Here, we present the X-ray crystal structure of Mpr1 and its complex with the substrate cis-4-hydroxy-L-proline at 1.9 and 2.3 Å resolution, respectively. Mpr1 is folded into α/β-structure with eight-stranded mixed β-sheets and six α-helices. The substrate binds to Asn135 and the backbone amide of Asn172 and Leu173, and the predicted acetyl-CoA-binding site is located near the backbone amide of Phe138 and the side chain of Asn178. Alanine substitution of Asn178, which can interact with the sulfur of acetyl-CoA, caused a large reduction in the apparent kcat value. The replacement of Asn135 led to a remarkable increase in the apparent Km value. These results indicate that Asn178 and Asn135 play an important role in catalysis and substrate recognition, respectively. Such a catalytic mechanism has not been reported in the GNAT proteins. Importantly, the amino acid substitutions in these residues increased the L-Δ(1)-pyrroline-5-carboxylate level in yeast cells exposed to heat stress, indicating that these residues are also crucial for its physiological functions. These studies provide some benefits of Mpr1 applications, such as the breeding of industrial yeasts and the development of antifungal drugs.
authors.affiliation: Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara 630-0192, Japan.
authors.forename: Ryo
authors.initials: R
authors.lastname: Nasuno
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Hirano
authors.forename: Takafumi
authors.initials: T
authors.lastname: Itoh
authors.forename: Toshio
authors.initials: T
authors.lastname: Hakoshima
authors.forename: Takao
authors.initials: T
authors.lastname: Hibi
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Takagi
mesh_terms: D000107:Acetylation; D000123:Acetyltransferases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D018360:Crystallography, X-Ray; D006909:Hydroxyproline / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D018384:Oxidative Stress / Q000235:genetics*; D011392:Proline / Q000378:metabolism*; D011487:Protein Conformation; D017510:Protein Folding; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
chemical_list: D029701:Saccharomyces cerevisiae Proteins; D011392:Proline; D000123:Acetyltransferases; C492447:Mpr1 protein, S cerevisiae; D006909:Hydroxyproline
keywords: X-ray crystallography; antioxidant enzyme; cyclic amine N-acetyltransferase; reaction mechanism